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MODIFIED HEPATOCYTES AND USES THEREFOR
Description
Background
The liver is of endodermal origin and the largest
05 gland in the human body. It has numerous crucial roles,
including bile secretion, participation in carbohydrate,
lipid and protein metabolism, fibrinogen production and
detoxification of drugs. The liver also serves as the
main site at which nutrients absorbed from the gastro-
10 intestinal tract and transported via the blood are
processed for use by other body cells.
Hepatocytes, which are the main type of parenchymal
or distinguishing cell in the liver, carry out the liver
functions and, thus, are responsible for synthesizing,
15 degrading and storing a wide variety of substances. In
addition, a system of small channels (canaliculi) and
larger ducts connects hepatocytes with the gut lumen.
Through this route, hepatocytes secrete bile, an emulsi-
fying agent which helps in absorption of ingested fats.
20 Hepatocytes are also the main location at which lipo-
protein particles for export are made; enzymes respons- -
ible for synthesis of the lipid constituents of lipo-
proteins occur in hepatocyte membranes.
/
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Because of the many important functions the liver
has, its inability to function normally (e.g., as a
result of a genetic defect or damage caused by alcohol or
other toxic substances) will often have significant
05 adverse effects on an individual's health. A means by
which normal function can be conferred upon or restored
to a liver whose function is compromised would be very
useful in treating, correcting or preventing such an
abnormality .
10 Summary of the Invention
The invention described herein relates to
genetically engineered or transduced hepatocytes which
express genetic material (DMA or RITA) of interest
introduced or incorporated into them, as well as to
15 methods of producing, transplanting and using the
genetically engineered hepatocytes. The genetic material
of interest can be incorporated through the use of a
vector, such as a recombinant retrovirus, which contains
the genetic material of interest, or by other means.
20 Hepatocytes of the present invention express the
genetic material of interest. Such genetic material of
interest can be: 1) genetic material present in and
expressed at biologically effective levels by normal
hepatocytes, but present in or expressed in less than
25 normal quantities in the hepatocytes prior to transfer of
genetic material of interest into them by the method of
the present invention; 2) genetic material not present in
normal hepatocytes; or 3) genetic material present in
normal hepatocytes but not expressed at biologically
30 effective levels in such cells, alone or in any
combination thereof.
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In hepatocytes of the present invention, the genetic
material of interest can be incorporated into the celluar
genetic material (e.g., into genomic DNA) or can be
present extrachromosomally (i.e., expressed episomally) .
05 The genetic material of interest can be DNA or RITA; the
DNA can constitute all or a portion of a gene of interest
(i.e., one whose expression in hepatocytes is desired).
The genetic material incorporated into and expressed
by hepatocytes of the present invention can additionally
10 include genetic material (e.g., DNA) encoding a
selectable marker, which provides a means by which cells
expressing the genetic material of interest are
identified and selected for. Hepatocytes containing
incorporated genetic material (i.e., genetic material of
15 interest and, optionally, genetic material encoding a
selectable marker) are referred to as transduced
hepatocytes.
Genetic material can be introduced into hepatocytes
ex vivo or in vivo . That is, it can be introduced, by
20 means of an appropriate vector, into isolated (cultured)
hepatocytes, which are subsequently transplanted into the
recipient. Alternatively, it can be introduced directly
into the recipient in such a manner that it is directed
to and taken up by target cells (hepatocytes) , where it
25 is incorporated and expressed. Particularly useful for
this purpose are retroviral vectors which have an
amphotropic host range and include the genetic material
of interest which is to be incorporated into hepatocytes.
Retroviral vectors have been used to stably
30 transduce hepatocytes with genetic material which
included genetic material encoding a polypeptide or
protein of interest and genetic material encoding a
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dominant selectable marker- Genetic material including
DNA encoding a polypeptide of interest and DNA encoding a
dominant selectable marker has been introduced into
cultured hepatocytes. Expression of the genetic material
by the hepatocytes into which they have been incorporated
has also been demonstrated.
A method of transplanting transduced hepatocytes
which express the incorporated genetic material they
contain is also a subject of the present invention.
Transduced hepatocytes of the present invention are used,
for example, for the delivery of polypeptides or proteins
which are useful in prevention and therapy of an acquired
or an inherited defect in hepatocyte (liver) function.
For example, they can be used to correct an inherited
deficiency of the low density lipoprotein receptor
(LDLR) , which is synthesized in hepatocytes, and to
correct an inherited deficiency of ornithine trans-
carbalyase (OTC) , which results in congenital hyper-
ammonemia.
Hepatocytes of the present invention are useful as a
means by which abnormal hepatocyte function can be
corrected. That is, hepatocytes can be transduced with
genetic material of interest selected to compensate for
over- or underproduction of a protein or peptide which is
synthesized correctly, but in inappropriate amounts in
the hepatocytes. Alternatively, they can be transduced
with genetic material of interest encoding a protein or
polypeptide which is produced in an appropriate quantity,
but is functionally defective (e.g., because of an
abnormal structure or amino acid makeup) .
Hepatocytes to be modified ex vivo , as described
herein, can be obtained from an individual, modified and
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returned to the individual by transplanting or grafting
or, alternatively, can be obtained from a donor (i.e., a
source other than the ultimate recipient) , modified and
applied to a recipient, again by transplanting or
05 grafting.
An important advantage of the procedure of the
present invention is that the genetically engineered
hepatocytes can be used to provide a desired therapeutic
protein or peptide by a means essentially the same as
10 that by which the protein or peptide is normally produced
and, in the case of autologous grafting, with little risk
of an immune response and graft rejection. In addition,
there is no need for extensive (and often costly) purifi-
cation of a polypeptide before it is administered to an
15 individual, as is generally necessary v/ith an isolated
polypeptide. Hepatocytes modified according to the
present invention produce the polypeptide as it would
normally be produced.
Because genes can be introduced into hepatocytes
20 using a retroviral vector, they can be "on" (subject to)
the retroviral vector * control; in such a case, the gene
of interest is transcribed from a retroviral promoter. A
promoter is a specific nucleotide sequence recognized by
RNA polymerase molecules that start RNA synthesis.
25 Alternatively, retroviral vectors having additional
promoter elements (in addition to the promoter
incorporated in the recombinant retrovirus) , which are
responsible for the transcription of the genetic material
of interest, can be used. For example, a construct in
30 which there is an additional promoter modulated by an
external factor or cue can be used, making it possible to
control the level of polypeptide being produced by the
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modified hepatocytes by activating that external factor
or cue. For example, heat shock proteins are proteins
encoded by genes in which the promoter is regulated by
temperature. The promoter of the gene which encodes the
metal-containing protein metallothionine is responsive to
cadmium (Cd ++ ) ions. Incorporation of this promoter or
another promoter influenced by external cues also makes
it possible to regulate the production of the polypeptide
by the engineered hepatocytes.
10 Brief Description of the Drawings
Figure 1 is a. schematic representation of a wild
type murine leukemia virxis (retroviral) genome.
Figure 2 is a schematic representation of retroviral
vectors f each having a recombinant genome, which are
15 useful in the present invention. Figure 2a is pLJ;
Figure 2b is pEm; and Figure 2c is pip.
Figure 3 is a schematic representation of vectors
which express human-LDLR. Each has a different trans-
criptional element which drives expression of LDLR:
20 LTR-LDLR - viral long term repeat sequences (LTR) ;
BA-LDLR - promoter from chicken beta-actin gene (BA) ;
H 4 -LDLR - promoter from human histone H4 gene (H4) ;
TK-LDLR - promoter from thymidine kinase gene of herpes
simplex virus (TK) .
25 Figure 4 is a three-day exposure of a Southern blot
in which the effect of extracellular matrix and the time
of infection on integration of provirus in rat hepatocyte
cultures are shown.
Figure 5 represents (panels A-F) cytochemical
localization of beta-galactosidase activity in transduced
cultures of rat hepatocytes and HIH3T3 cell and (panels
30
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G-K) liver- specif ic cytochemical and immunocytochemical
stains of rat hepatocyte cultures.
Figure 6 presents results of a Southern analysis of
transduced rabbit hepatocytes.
05 Figure 7 presents results of a Northern analysis of
transduced rabbit hepatocytes.
Figure 8 is a graphic representation of human
parathyroid hormone (PTH) production by transduced rat
hepatocytes and of rat albumin production by control
10 hepatocytes.
Detailed Description of the Invention
The present invention is based on development of an
effective method of introducing genetic material of
interest into hepatocytes and of a method of
15 transplanting hepatocytes containing the genetic material
of interest. Using an appropriate vector, such as a
retroviral vector, which includes the genetic material of
interest, or other means, it is possible to introduce
such genetic material into hepatocytes , where it is
20 expressed. In particular, it has been demonstrated that
DNA of interest can be efficiently and stably introduced
into mature cultured hepatocytes, which subsequently
express the DNA (produce the encoded polypeptide) , and
that the transduced hepatocytes can be grafted or
25 transplanted. In addition, such a vector can be used to
introduce genetic material of interest into hepatocytes
in vivo , thus avoiding the need to transplant or graft
transduced hepatocytes.
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The following is an explanation of isolation of
hepatocytes to be transduced ex vivo and grafted or
transplanted into a recipient, using the method of the
present invention; of vectors useful in introducing
05 genetic material of interest into hepatocytes , either ex
vivo or in vivo ; and of the method of the present
invention by which hepatocytes are transduced*
Isolation of Hepatocytes
In one embodiment of the method of the present
10 invention, cultured mature hepatocytes are transduced and
subsequently grafted or transplanted into a recipient.
In this embodiment, hepatocytes are obtained, either from
the recipient (i.e., the individual who is to receive the
transduced hepatocytes) or from a donor, using known
15 techniques. In general, this includes removing all or a
portion of a liver, from which hepatocytes are removed by
in situ perfusion of a collagenase solution. For
example, in the case of isolation of hepatocytes from an
intact liver, a catheter is inserted into a vein which
20 either leaves or enters the liver, collagenase solution
is perfused through and hepatocytes are released. In the
case of a liver biopsy, which results in a cut or exposed
surface, a small catheter (or catheters) is inserted into
vessels on the open or cut surface. Collagenase solution
25 is perfused through the catheterized vessels, resulting
in release of hepatocytes. Once removed or isolated, the
hepatocytes are plated and maintained under conditions
(e.g., on appropriate medium, at correct temperature,
etc.) suitable for transfection.
30 For example, several methods have been described for
isolating highly enriched populations of rat hepatocytes
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and maintaining these cells in culture for extended
periods of time. Koch f K.S. and H.L. Leffert, Annals
IT. Y. Academy of Sciences , 349 : 111-127 (1980); McGowan,
J. A. et aL f Journal of Cellular Physiology , 108 : 353-363
05 (1981)? Bissell, D.M. and P.S. Guzelian, Annals of the
New York Academy of Sciences , 349:85-98 (1981); and Enat f
R. et al. , Proceedings of the National Academy of
Sciences, U.S.A. , £1:1411-1415 (1984). Such methods can
be used to isolate and maintain hepatocytes to be
10 transduced by the method of the present invention.
Hepatocytes can also be prepared using a modification of
the procedure developed by Barry and Friend, described
below and in Example 1, with the perfusion mixture
described by Leffert. Leffert, H.L. et al. , Methods in
15 Enzymology , 513:536-544 (1979), the teachings of which are
incorporated herein by reference.
Retroviral Vectors
One limitation of hepatocyte cultures for studying
the molecular aspects of processes such as gene regula-
20 tion has historically been the lack of efficient gene
transfer techniques. Conventional methods of transfec-
tion are inefficient and toxic to the cells. Tur-Kaspa,
r. et al. , Molecular and Cellular Biology , 6_:716-718
(1986) . As described below, recombinant retroviruses
25 have been used to overcome these problems. As a result,
it is now possible to efficiently and stably transduce
primary cultures of hepatocytes by replication-defective
retroviruses. Such replication-defective retroviruses
have been used to introduce genetic material of interest
30 into cultured hepatocytes. Transduction is efficient and
produces hepatocytes which express the genetic material
of interest (i.e., produce the encoded protein or
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polypeptide) and which retain the ability to be
tr an sp lanted .
Retroviruses are RNA viruses; that is r the viral
genome is RNA. This genomic RNA is, however, reverse
055 transcribed into a DNA intermediate which is integrated
very efficiently into the chromosomal DNA of infected
cells. This integrated DNA intermediate is referred to
as a provirus. As shown in Figure l r the retroviral
genome and the proviral DNA have three genes: the gag .
Iff; the pol and the env, which are flanked by two long
terminal repeat (LTR) sequences. The gag gene encodes
the internal structural (nucleocapsid) proteins; the pol
gene encodes the RNA-directed DNA polymerase (reverse
transcriptase) ; and the env gene encodes viral envelope
15 glycoproteins. The 5' and 3 f LTRs serve to promote
transcription and polyadenylatior. of virion RNAs.
Adjacent to the 5 1 LTR are sequences necessary for
reverse transcription of the genome (the tRHA primer
binding site) and for efficient encapsidation of viral
20 RNA into particles (the Psi site) „ Mulligan, R.C. , In:
Experimental Manipulation of Gene Expression , M . Inouye
(ed> r 155-173 (1983); Mann, R. , et al. , Cell , 33:153-159
(1983); Cone, R.D . and R.C* Mulligan, Proceedings of the
National Academy of Sciences, U.S.A. , 81^:6349-6353
25 (1984) .
If the sequences necessary for encapsidation (or
packaging of retroviral RNA into infectious virions) are
missing from the viral genome, the result is a cis defect
which prevents encapsidation of genomic RNA. However,
30 the resulting mutant is still capable of directing the
synthesis of all virion proteins. Mulligan and co-
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workers have described retroviral genomes from which
these Psi sequences have been deleted, as well as cell
lines containing the mutant stably integrated into the
chromosome. Mulligan, R.C., In: Experimental Manipu-
05 lation of Gene Expression , M. Inouye (ed) , 155-173
(1983); Mann, R. , et al. , Cell , 32:153-159 (1983); Cone,
R.D. and R.C. Mulligan, Proceedings of the National
Academy of Sciences, U.S.A. , 8_1: 6349-6353 (1984). The
teachings of these publications are incorporated herein
10 by reference.
The Psi 2 cell line described by Mulligan and
co-workers was created by trans fecting NIH 3T3 fibro-
blasts with pMOV-Psi", which is an ecotropic Moloney
murine leukemia virus (Mo-MuLV) clone. pMOV-Psi" expres-
15 ses all the viral gene products but lacks the Psi se-
quence, which is necessary for encapsidation of the viral
genome. pMOV-Psi" expresses an ecotropic viral envelope
glycoprotein which recognises a receptor present only on
mouse (and closely related rodent) cells.
20 Another cell line is the Psi am line, which are
Psi-2-like packaging cell lines. These Psi-am cell lines
contain a modified pMOV-P si-genome, in which the eco-
tropic envelope glycoprotein has been replaced with
envelope sequences derived from the amphotropic. virus
25 4070A. Hartley, J.W. and W.P. Rowe, Journal of Virology ,
L9:19-25 (1976). As a result, they are useful for
production of recombinant virus with a broad mammalian
host range, amphotropic host range. The retrovirus used
to make the Psi am cell line has an amphotropic host
30 range and can be used to infect human cells. If the
recombinant genome has the Psi packaging sequence, the
Psi-am cell line is capable of packaging recombinant
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retroviral genomes into infectious retroviral particles.
Cone, R. and R. Mulligan, Proceedings of the National
Academy of Sciences, USA f jTl: 6349-6353 (1984).
The retroviral genome has been modified by Cone and
OS Mulligan for use as a vector capable of introducing new
genes into cells. As shown in Figure 2, the gag , the pol
and the env genes have all been removed and a DNA segment
encoding the neo gene has been inserted in their place.
The neo gene serves as a dominant selectable marker. The
10 retroviral sequence which remains part of the recombinant
genome includes the LTRs, the tRNA binding site and the
Psi packaging site. Cepko, C. et al. , Cell , 37:1053-1062
(1984) .
Additional vector constructions which can be used in
15 producing transduced hepatocytes of the present invention -
are represented in Figure 2 and are described in detail
below.
pLJ . The characteristics of this vector have been
described in Korman, A.J. et al. , Proceedings of the
20 National Academy of Sciences, USA , 8£:2150 (1987). This
vector is capable of expressing both the gene of interest
and a dominant selectable marker, such as the neo gene.
The gene of interest is cloned in direct orientation into
a BamHI/Smal/Sall cloning site just distal to the 5 f LTR,
25 while the Neo gene is placed distal to an internal
promoter (from SV40) which is farther 3' than is the
cloning site (is located 3 * of the cloning site) .
Transcription from pLJ is initiated at two sites: 1) the
5' LTR, which is responsible for expression of the gene
30 'of interest and 2) the internal SV40 promoter, which is
responsible for expression of the neo gene. The struc-
ture of pLJ is represented in Figure 2a.
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Vector pLJ is represented in Figure 2a. In pL J , the
genetic material of interest is inserted just following
the 5' LTR. Expression of this genetic material is
transcribed from the LTR and expression of the neo gene
05 is transcribed from an internal SV40 promoter.
pEM . In this simple vector, the entire coding
sequence for gag , pol and env of the wild type virus is
replaced with the gene of interest, which is the only
gene expressed. The components of the pEIl vector are
10 described below. The 5' flanking sequence, 5 1 LTR and
40 0 bp of contiguous sequence (up to the Bara HI site) is
from pZIP. The 3' flanking sequence and LTR are also
from pZIP; however, the Clal site 150 bp upstream from
the 3' LTR has been ligated with synthetic BamH I linkers
15 and forms the other half of the Bam HI cloning site
present in the vector. The Hindlll/EcoRI fragment of
pBR322 forms the plasmid backbone. This vector is
derived from sequences cloned from a strain of Moloney
Murine Leukemia virus. An analogous vector has been
20 constructed from sequences derived from the
myeloproliferative sarcoma virus. The structure of pEM
is represented in Figure 2b.
pip . This vector is capable of expressing a single
gene driven from an internal promoter. The structure of
25 pip is represented in Figure 2c. The construction of
these vectors is summarized below. The 5 f section of the
vector, including the 5' flanking sequences, 5 1 LTR, and
1400 bp of contiguous sequence (up to the xho site in the
qag region) is derived from wild type Moloney Leukemia
30 virus sequence. Shinnick et al. , Nature , 293:543 (1918).
The difference between the two is that a SacII linker is
cloned into an Haelll restriction site immediately
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adjacent to the ATG of the gag gene. The 3 r section of
the vector, including the 3' flanking sequences, 3 1 LTR
and 3 1 contiguous sequence (up to the cla l site in the
env coding region) is from pZIP. However, there are two
05 modifications: 1) the cla l site has been linked to BamH I
and 2) a small sequence in the 3 f LTR spanning the
enhancer (from PvuII to Xbal) has been deleted. Bridging
the 5* and 3' sections of the vector is one of several
promoters; each one is contained on a xhoI/BamHI
10. fragment, and each is capable of high level constitutive
expression in most tissues. These promoters include
beta-actin from chicken (Chcudory, P.V. et ah , CSH
Symposia Quantitative Biology , L.I. 1047 (1986), and
thynidine kinase from Herpes Simplex Virus, histone H4
15 from human (Hanly, S.I1. et al. , Moleciilar and Cellular
Biolocry , 5^:380 (1985)). The vector backbone is the
Hindlll/EcoRI fragment from pBR322. The gene of interest
is cloned into the Barafll site in direct orientation, just
downstream from the internal promoter.
20 Vectors without a selectable marker can also be used
to transduce endothelial cells with genetic material of
interest. Such vectors are basically simplifications of
the vectors previously described, in which there is such
a marker. Vector pE£l is represented in Figure 2b; as
25 represented, the main components of the vector are the 5*
and 3 1 LTR, and the genetic material of interest, in-
serted between the two LTRs.
Retroviral vectors useful for ex vivo modification of
hepatocvtes
30 Four additional recombinant retroviruses suitable
for introducing genetic material of interest into
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cultured hepatocytes and expressing biologically
significant amounts of the encoded protein or polypeptide
are also represented in Figure 3, with specific reference
to the human LDLR gene. These vectors, which included
05 the human LDLR gene, have been used to efficiently
transduce hepatocytes, which then expressed levels of
LDLR equal to normal endogenous levels. These vectors
will be illustrated with reference to the LDLR gene, but
any nucleotide sequence of interest can be incorporated
10 into the retroviruses and introduced into hepatocytes.
As shown, each vector differs in the transcriptional
elements used to drive expression of the gene. These are
described in detail in Example IV. Briefly, in the
vector LTR- LDLR, transcription should begin at the 5'
15 LTR, with the result that a single full length viral
transcript, in this case, one which expresses LDLR, is
produced. In the remaining vectors, expression of LDLR
should be driven by transcripts initiated* from trans-
criptional control sequences located internal to the
20 proviral transcriptional unit.
Each of these latter vectors differs in the
transcriptional elements responsible for transcription:
BA-LDLR contains the promoter from chicken beta-actin
gene? H4-LDLR contains the promoter from the human
25 histone H4 gene; and TK-LDLR contains the promoter from
the thymidine kinase gene from herpes simplex virus.
Each of the three vectors also contains a deletion of
viral transcriptional enhancer sequences located in the
3' LTR, in order to reduce the amount of viral
30 transcription which occurs after reverse transcription
and integration of the recombinant provirus. Human LDLR
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coding sequences for all four vectors were derived from a
full length human LDLR cDNA insert.
As described in Example IV, use of these vectors in
transducing hepatocytes resulted in levels of viral-
05 directed LDLR RNA in the transduced cells that exceeded
endogenous levels (50-100X) . The amount of functional
receptor produced, however, was less than or equal to
normal endogenous levels for most vectors, possibly due
to insufficient RNA processing or transport or diminished
10 translational efficiency.
Because a potential problem with the ex vivo method
of hepatocyte modification is the fact that only a
fraction of the total hepatocytes isolated can be
engrafted , additional retroviral vectors that express
15 higher levels of the encoded protein (here, LDLR) may be
useful. Such vectors may be useful, for example, as a
means of decreasing the number of transplanted cells
required for production of useful quantities of the
encoded protein. For example, each vector can include
20 specialized transcriptional control sequences which are
internal to the proviral transcriptional unit. This
makes it possible to provide variation in the level of
transcription of the nucleotide sequence of interest. Of
particular interest here are transcriptional elements
25 from genes expressed at high levels in liver cells (e.g.,
alpha-fetoprotein, albumin) .
To enhance the translational efficiency of the
chimeric nucleotide sequence of interest RNAs generated
by the vectors, (e.g., by generating more translatable
30 RNAs) , selected 5* and 3' nontranslated sequences from a
well-characterized gene (e.g., RU-5 region of a human
retrovirus) or the authentic 5' and 3' nontranslated
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sequences of the nucleotide sequence of interest can also
be included in the retroviral vectors used.
Because it is possible that constitutive production
of very high levels of the encoded protein (e.g., LDLR)
05 may result in toxicity to cells, it may also be
appropriate to include in the vector sequences from the
nucleotide sequence of interest (or gene) which confer
transcriptionally-mediated end-product repression. For
■ example, in the case of the LDLR gene, sequences (called
10 the steroid responsive elements) which confer
transcriptionally-mediated, end-product repression by
sterols can be included in the vector.
Retroviral vectors for in vivo modification of
hepatocvtes
15 It is also possible to use vectors, particularly
recombinant retroviral vectors, to transduce hepatocyte
cells in vivo . For example, one strategy for targeting
the LDLR gene to hepatocvtes can be based on the presence
of the asialoglycoprotein (ASGP) receptor on hepatocytes.
20 This receptor, which is specifically expressed in
hepatocytes f is involved in the uptake and catabolism of
glycoproteins that have had their terminal sialic acids
removed, thereby exposing penultimate galactose residues.
Glycoprotein-receptor complexes are internalized by
25 receptor mediated endocytosis. Asialoglycopeptide-
protein conjugates and asialoglycopeptide-coated vesicles
have been used to specifically deliver a variety of
bioactive agents to the liver in vivo . Aiti§, A.D. et
al. , Proceedings of the National Academy of Sciences,
30 USA, 27:5923-5927 (1980); Fiumw, L. et al. , FEES Letter ,
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0£
10:
15
20
25'
PCT/US89/00422
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103 :47-51 (1979); Hildenbrandt, G.R. and N.N. Aronson,
BBA, £31:499-502 (1980).
For example, one approach to targeting delivery of
genes to hepatocytes can be based on the modification of
existing retroviruses to make them ligands for the ASGP
receptors. The envelope proteins from murine leukemia
viruses are complex glycoproteins that have a high
content of sialic acid. Internalization of virus occurs
through the specific interaction of the viral envelope
with" a cell surface receptor, followed by receptor-
mediated endocytosis of the virus /receptor complex.
Hepatocyte- specific transduction may be possible if
modified virions that are specifically internalized by
the AGSP receptor are developed and used. For example,
it is possible to use viruses whose envelope protein is
devoid of sialic acid r thereby rendering them ligands for
the AGSP receptor. One approach is to enzymatically
remove the terminal sialic acids from intact virions with
neuraminidase. Alternatively, it is possible to con-
struct viral producer lines with genetically modified
envelope genes that code for glycoproteins with terminal
galactose residues. It is possible to construct chimeric
envelope genes that encode fusion proteins in which the
carboxy terminal sequences are derived from the 3 1
portion of the envelope gene and the amino terminal
sequences are derived from genes of known ligands for
ASGP receptor. In addition, it is possible to use
lectin-resi stance selection systems to isolate mutants of
the viral producer lines that are incapable of adding
terminal sialic acids to N-linked chains. . Viruses
produced from these lines should bind to the ASGP
receptor. Transduction of cells that express ASGP
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receptor with these modified virions can be tested in
vitro and in vivo .
Another approach to the targeted delivery of genes
to hepatocytes has recently been described by Wu. Wu,
05 G.Y. and C.H. Wu, Journal of Biological Chemistry /
2_62:4429-4432 (1987). Wu coupled pSV2CAT plasmid to a
ligand of the ASGP receptor called asialoorosomucoid
(ASOR) and demonstrated in HEP G2 cells uptake of the
conjugates via the ASGP receptor and expression of CAT
10 activity. After administration of this conjugate to
rats, specific, but transient, expression of CAT was
demonstrated in liver homogenates. It is possible that
this method can be modified to produce a method by which
recombinant retroviral vectors or episomal vectors can be
15 introduced into hepatocytes in vivo . The efficiency and -
specificity of this delivery system can be assessed, for
example, by transferring an expression vector that stably
expresses a product which can be detected in situ by
direct analysis of the product (e.g., beta-galactosi-
20 dase) .
Successful application of in vivo targeting requires
stable expression of the transferred gene. Transduction
of hepatocytes in vivo with retroviruses that have been
modified to allow for uptake via the ASGP receptor will
25 require integration of the provirus into genomic DNA.
Integration is rare in the quiescent liver, however. The
efficiency of retroviral integration may be improved by
exposing the recipient to virus following partial
hepatectomy. During this time, the residual hepatocytes
30 undergo rapid proliferation. This, however, may not be
practical clinically. One way to avoid this problem is
to modify the retroviral vector* in such a way as to
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promote its persistence as an episome in the fern of a
double stranded DNA circle. This can be done by
incorporating the cis (oriP) and trans (EBNA) sequences
of Epstein Barr Virus that are required for plasmid
05; replication into a retrovirus vector. It is also
possible to use other sequences (called autonomously
replicating sequences) isolated from the eukaryotic
(e,g. r mouse) genome which have been shown to drive the
autonomous replication of plasmids. In these cases,
Iff vectors containing deletions of sequences within the
inverted repeats of the long terminal repeats which
prevent proviral integration into the host chromosome,
will be used.
Introduction of Genetic Material into Hepatocvtes
15 Genetic material of interest has been incorporated
into cultured hepatocytes and expressed in the resulting
genetically engineered hepatocytes, as described below
and in the examples.
Genetic material which can be incorporated into .
20 hepatocytes according to the method described can be:
1) genetic material (DNA or RNA) which is present in and
expressed at biologically effective levels (levels
sufficient to produce the normal physiological effects of
the polypeptide it encodes) in normal hepatocytes, but
Z5 present in or expressed in less than normal quantities in
the hepatocytes prior to stable transfer of genetic
material of interest into them by the method of the
present invention; 2) genetic material not present in
normal hepatocytes; or 3) genetic material present in
30 normal hepatocytes but not expressed at biologically
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effective levels in such cells, alone or in any combina-
tion thereof.
The genetic material incorporated into and expressed
by hepatocytes can also, optionally, include genetic
05 material encoding a selectable marker, thus making it
possible to identify and select cells which contain and
express the genetic material of interest.
Thus, DNA or RNA introduced into cultured
hepatocytes of the present invention includes the genetic
10 material (DNA or RNA) of interest and, optionally,
genetic material encoding a selectable marker; such DNA
or RNA is referred to as incorporated genetic material
(or incorporated DNA, incorporated RNA) . Hepatocytes
containing incorporated genetic material are referred to
15 as transduced hepatocytes; they express the DNA or RNA of.
interest and produce the encoded protein or polypeptide.
Exogenous DNA encoding a polypeptide or protein of
interest and f optionally, a selectable marker (e.g., neo,
. which encodes neomycin resistance) is incorporated in
20 vitro into hepatocytes as described below and in Examples
I-III. Hepatocytes isolated as described previously are
plated at subconfluent density on matrix substrata and
maintained in hormonally defined media, such as that
described by Enat et al. , the teachings of which are
25 incorporated herein by reference. Enat, R. f et al. ,
Proceedings of the National Academy of Sciences, USA ,
81:1411-1415 (1984). The media is changed as needed to
maintain the hepatocytes.
Cells are subsequently infected with an amphotropic
30 retrovirus which contains DNA of interest (e.g., DNA
encoding a. polypeptide whose expression in hepatocytes is
desired) and, optionally, DNA encoding a selectable
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marker to be incorporated into the hepatocytes. The
hepatocytes are infected with the recombinant retrovirus
(and thus transduced with the DNA of interest) by expos-
ing them to virus which has a recombinant genome. This
05 results in infection of the cells by the recombinant
retrovirus. It is possible to optimize the conditions
for infection of the hepatocytes by using a high titer
amphotropic virus.
A cell line which produces recombinant anphotropic
10 retrovirus having a recombinant genome is used to infect
hepatocytes. The recombinant genome can include a
variety of components, but in general is comprised of two
LTRs and, in place of the gag , the pol and the env
sequences, a second promoter sequence; in some cases, it
15 also includes a gene encoding a selectable marker (e.g., -
neo) .
Viral stocks, to be used in introducing genetic
material of interest into hepatocytes, are harvested, as
described above, supplemented with Polybrene (Aldrich)
20 and added to the culture of hepatocytes. If the titer of
the virus is high (e.g., approximately 10** cfu per ml.),
then virtually all hepatocytes will be infected and no
selection of transduced hepatocytes is required. If the
titer is very low, then it is necessary to use a
25 retroviral vector that has a selectable marker, such as
neo or his. If a selectable marker is used, after
exposure to the virus, the cells are grown to confluence
and split into selective media (e.g., media containing
G418 if the selectable marker is neo, media containing
30 histidinol and no histidine if the selectable marker is
his) .
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The neo gene is a bacterial gene derived from the
transposon Tn5 , which encodes neomycin resistance in
bacteria and resistance to the antibiotic G418 in mamma-
lian cells. This neo gene acts as a dominant selectable
05 marker; its presence in a mammalian cell converts the
cell into one which will grow in the presence of G418.
(If it is not present, the cell dies in the presence of
G418.) As a result, the presence of this gene in a
mammalian cell can be determined by culturing cells in
10 media which contains G418.
The recombinant retroviral vectors having the neo
gene also have a cloning site. As a result, genetic
material of interest can be introduced into the vector,
incorporated into hepatocytes and expressed by hepato-
15 cytes transduced with the recombinant retrovirus
(referred to as hepatocytes containing incorporated
genetic material) . At the Bam HI cloning site, it is
possible to. insert genetic material of interest. As
described above, hepatocytes have been transduced with
20 the gene encoding beta-galactosidase from S , coli . The
efficiency of transduction was assessed, as described in
Example III. Expression of the beta-galactosidase gene
was also assessed and is detailed in Example III.
For example, a helper-free amphotropic producer is
25 grown in tissue culture to a confluent density in
Dulbecco's Modified Eagle f s Medium (DME) supplemented
with 10% calf serum (CS) . Fresh media is added and
subsequently the media is harvested. The spent media
(or viral stock) is filtered to remove detached producer
30 cells and is used immediately to infect cells or is
stored (e.g., at -70°C) for later use.
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Media is removed from a subconf luent plate of
hepatocytes (recipient hepatocytes) and quickly replaced
with viral stock which contains Polybrene (Aldrich) .
Subsequently, this is removed and replaced with fresh
Q5 media * Thus, the media used is a viral supernatant and
the recombinant genome of the infectious virus includes
DNA of interest. The infection procedure results in
hepatocytes which express the DHA encoding a gene product
of interest and, optionally , a selectable marker.
10 In one embodiment, hepatocytes are exposed to media
containing infectious virus produced in Psi am cells; the
infectious virus contain a recombinant genome having the
genetic material of interest. The recombinant genome in
one instance includes genetic material encoding a protein
15 or a polypeptide (e.g., the receptor for low density
lipoproteins; ornithine transcarba lyase) and, optionally,
a gene encoding a dominant selectable marker (e.g., the
r.eo gene which encodes neomycin resistance) . As a
result, the hepatocytes are transduced — that is, the
20 genetic material of interest (for example, DNA encoding a
polypeptide or a protein of interest) and, optionally,
the neo gene are stably introduced into the hepatocytes.
The transduced hepatocytes express the encoded protein or
polypeptide and, if the neo gene is present, express it,
25 resulting in cells having the selectable trait.
One embodiment of the present invention is described
in detail in the Examples. Briefly, hepatocytes were
prepared using a modification of the method of Berry and
Friend with the perfusion mixture of Leffert. Berry ,
30 M.N. et al. , J. Cell. Biol. , 43:506-520 (1969); Leffert,
H.L. et al., Methods Enzvmol. , 58:536-644 (1979). The
resulting hepatocytes were plated at a density of 3-4x10
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2
cells/cm onto Primaria plates substratum in horaionally
defined media supplemented with 10% fetal bovine serum.
The media was replaced with fresh hormonally defined
media, which was subsequently changed periodically .
05 Spent media was obtained by harvesting DME media
supplemented with 10% calf serum in which a helper-free
amphotropic producer of the BAG virus was cultured;
Conditions for infection of the hepatocytes were
optimized through the use of BAG, which is a high titer
10 amphotropic virus encoding E. coli beta galactosidase .
The producer coexpressed beta-galactosidase from E, coli
and the bacterial neo gene. The spent media was filtered
to remove detached producer cells and used as viral stock
to infect hepatocytes.
15 Cells were infected by removing media from a sub-
confluent plate of hepatocytes (recipient hepatocytes)
and replacing it with viral stock. Hepatocyte cultures
were infected in this way for approximately 12 hours.
Hepatocytes containing the DNA of interest (i.e., DNA
20 encoding beta-galactosidase) and the neo gene were
isolated by culturing on media containing the antibiotic
G418. Those into which the recombinant retrovirus was
effectively introduced by infection, referred to as
transduced hepatocytes, produce beta-galactosidase and
25 are neomycin resistant. The ability of hepatocytes
transduced with the recombinant retrovirus having the
beta-galactosidase gene to produce beta-galactosidase has
been assessed in vitro . This assessement is described in
Example III.
30 Rat hepatocytes have also been transduced with the
gene encoding human parathyroid hormone (hPTH) and shown
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to express the encoded hormone. This is described in
Example I and results are presented in Figure 8.
As a result, it has been demonstrated that trans-
duced hepatocytes secrete a polypeptide (beta-
05 galactosidase) which is normally not secreted by
hepatocytes. A similar approach can be used to introduce
any genetic material into hepatocytes in such a manner
that the encoded product is made and to assess expression
of the incorporated genetic material.
10 For example, hepatocytes have been transduced as a
result of exposure to media containing infectious virus
in which the recombinant genome includes DNA encoding
human LDLR. This is described in detail in Example IV.
Hepatocytes were infected with four LDLR virus prepara-
15 tions (each including one of the vectors represented in
Figure 3) and subsequently analyzed for gene transfer and
LDLR expression.
The same vectors can be used in the method of the
present invention, to introduce into cultured hepatocytes
20 any nucleotide sequence or gene of interest. Alterna-
tively, other vectors can be used, as can other means of
introducing genetic material of interest into cultured
hepatocytes .
As explained previously, it may also be possible to
25 introduce genetic material of interest into hepatocytes
in vivo . As described, a recombinant retrovirus in which
the genome includes the genetic material of interest
would be targeted to hepatocytes, with the result that
after introduction into an individual's body (e.g., by
30 intravenous injection) , the retrovirus is specifically
taken up by hepatocytes. Once taken up by hepatocytes,
the recombinant retroviral genome will be expressed,
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resulting in production of the protein or polypeptide
encoded by the genetic material of interest. Preferably,
the virus used to introduce the genetic material of
interest is modified (e.g., through inclusion of
as sequences, referred to as autonomously replicating
sequences or ARS) in such a manner that integration of
the provirus into the host chromosomes does not occur.
As a result, replication will occur episomally.
Use of Dominant Selectable Markers in the Introduction of
10 Genetic Material Encoding Polypeptides
In addition to genetic material of interest, a
vector can include genetic material encoding a selectable
marker, the presence of which makes it possible to
identify and select for cells transduced with the genetic -
15 material of interest. As described previously and in
Example III, the neo gene, which is such a marker, has
been used for this purpose. It is also possible to use
dominant selectable markers other than the neo gene to
introduce genetic material into hepatocytes. For
20 example, the His D- gene can be used for this purpose.
The His D gene is a bacterial gene from Salmonella and
encodes histidinol dehydrogenase, a polypeptide which
converts histidinol to histidine. Histidine is an
essential amino acid; histidinol is an alcohol analogue
25 of histidine and can be converted to histidine under the
proper metabolic conditions. If cells are grown in media
containing histidinol but lacking histidine, those cells
having the His D gene can convert histidinol to
histidine. Because histidine is essential to their
30 function, those cells which have the His D gene (and thus
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can make histidine) will survive and those lacking the
gene will not.
A retrovirus vector having the His D gene has been
used to infect keratinocytes. The keratinocytes contain-
05? ing His D gene were selected by growing these cells in
media lacking histidine but containing histidinol. As
expected, keratinocytes having the His D gene formed
colonies and grew to confluence; those lacking the gene
did not. In fact, such cells occurred at a much higher
ID? frequency than those in which the neo gene was included.
These same techniques are useful in selecting fibroblasts
containing DNA of interest.
As a result of this work, it is also possible to use
independent dominant selectable markers (e.g., the neo
15 gene and the His D gene) to introduce more than one type
of new genetic material into hepatocytes. In the case of
gene products which have two different subunits, for
example, separate dominant selectable markers can be used
to introduce the genetic information encoding the two
2Q subunits. In addition, two or more dominant selectable
markers can be used in the case of gene products which
need to be specifically cleaved or processed in order to
become active. A gene encoding the necessary processing
enzyme can be introduced along with the gene encoding the
25 polypeptide requiring such processing. This would enable
hepatocytes to process the polypeptide hormone.
Other Vehicles for the Introduction of Genetic Material
of Interest into Hepatocytes
It is also possible to use vehicles other than
30 retroviruses to genetically engineer or modify
hepatocytes. Genetic information of interest can be
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introduced by means of any virus which can express the
new genetic material in such cells. For example, SV40,
herpes virus, adenovirus and human papilloma virus can be
used for this purpose. DNA viruses can also be used to
05 introduce genetic material of interest, as well as a gene
encoding a selectable marker, into hepatocytes according
to the method of the present invention.
Transplantation of Transduced Hepatocytes
Hepatocytes expressing the incorporated genetic
10 material are grown to confluence in tissue culture
vessels; removed from the culture vessel; and introduced
into the body. This can be done surgically, for example.
In this case, the tissue which is made up of transduced
hepatocytes capable of expressing the nucleotide sequence
15 of interest is grafted or transplanted into the body.
For example, it can be placed in the abdominal cavity in
contact with/grafted onto the liver or in close proximity
to the liver. Alternatively, the transduced hepatocyte-
containing tissue can be attached to microcarrier beads,
20 which are introduced (e.g., by injection) into the
peritoneal space of the recipient. This approach has
been shown to be successful by transplantation of wild
type hepatocytes into a strain of rats (Nagase
analbuminemic rats) which are deficient in albumin
25 synthesis and demonstration of moderate levels of albumin
in serum of transplanted animals. Direct injection of
genetically modified hepatocytes into the liver may also
be possible.
Once introduced into the body of an individual, the
30 transduced hepatocytes provide a continuous supply of the
hormone, enzyme or drug encoded by the genetic material
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of interest. The amount of the hormone, enzyme or drug
supplied in this way can be modified or regulated as
needed (e.g., by using external cues or factors which
control or affect production, by controlling the size of
05 the graft or the quantity of fibroblasts introduced into
the body, or by removing the graft) .
Genetically modified hepatocytes have been implanted
into WHHL rabbits which lack LDL receptor function.
Hepatocytes icere isolated and infected with the LTR-LDLR
ICT- recombinant retrovirus. The transfected hepatocytes were
harvested and injected into the portal vein of WHHL
rabbits. Results demonstrated that serum cholesterol
levels decreased 30% over a 3 -day period, suggesting that
it is possible to at least partially correct the
15 metabolic abnormalities in the WHHL rabbit by
transplantation of genetically modified hepatocytes.
Uses of Genetically Modified Hepatocytes Having In-
corporated Genetic Material
The present invention makes it possible to geneti-
20 cally engineer hepatocytes in such a manner that they
produce a gene product (e.g., a polypeptide or a protein)
of interest in biologically significant amounts. The
hepatocytes formed in this way can serve as a continuous
drug delivery system to replace present regimens, which
25 require periodic administration (by ingestion, injection,
etc.) of the needed substance.
Incorporation of genetic material of interest into
hepatocytes would be particularly valuable in the treat-
ment of inherited disease and the treatment of acquired
30 disease. In the case of inherited diseases, this
approach is used to provide genetically modified
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hepatocytes which contain DNA encoding a protein or
polypeptide which an individual's hepatocytes are unable
to make correctly. For example, this could be used in
treating urea cycle disorders* Hepatocytes of the
05 present invention can also be used in the treatment of
genetic diseases in which a product (e.g., LDL receptor)
normally produced by the liver is not produced or is made
in insufficient quantities. Here, hepatocytes transduced
with a DNA encoding the missing or inadequately produced
10 substance can be used to produce it in sufficient quanti-
ties. In this case, the transduced hepatocytes would
produce LDL receptor and thus provide a means of
preventing or treating familial hypercholesterolemia,
which is an inherited disease in which the primary
15 genetic defect is an abnormality in the expression or
function of the receptor for low density lipoproteins.
This leads to elevated levels of serum cholesterol and
the premature development of coronary artery disease.
The transduced hepatocytes could be used to produce
20 sufficient quantities of the LDL receptor to overcome the
underlying defect.
There are also acquired diseases for which treatment
can be provided through use of genetically engineered
hepatocytes. For example, such cells can be used in
25 treating the coagulopathy associated with liver failure.
In this case, hepatocytes having incorporated in them a
gene encoding one or more clotting factors would correct
the acquired deficiency of these factors which leads to
bleeding. It may also be possible to treat viral
30 hepatitis, particularly hepatitis B or nonA-nonB
hepatitis, by gene transfer. For example, using the
method of the present invention, a gene encoding an
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anti- sense gene could be introduced into hepatocytes to
inhibit viral replication. In this case, a vector
including a structural hepatitis gene in the reverse or
opposite orientation would be introduced into
05 hepatocytes, resulting in production in transduced
hepatocytes of an anti-sense gene having the correct
orientation.
The present invention will now be illustrated by the
following examples, which are not to be seen as limiting
10 in any way.
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EXAMPLE I Hepatocyte Isolation and Culture
Rat hepatocytes were prepared by the procedure of
Berry and Friend, using the perfusion mixture of Leffert.
Berry, M. N. and D. S. Friend, Journal of Cell Biology,
05 43:506-520 (1969); Leffert, H. L. et al. , Methods in
Enzvmology , 58:536-544 (1979). Male Sprague-Dawley rats
weighing between 200 and 250 gms were used as the source
of hepatocytes. Cells were plated at a density of 4 x
10 4 cells/cm 2 onto one of several matrix substrata in
10 hormonally defined media supplemented with 10% fetal
bovine serum. Enat, R. et al. , Proceedings of the
National Academy of Sciences, USA , 81:1411-1415 (1984).
Four hours later the media was replaced with fresh
hormonally defined media which was subsequently changed
15 every 24 hours during the duration of the experiment.
The following substrata were used: 1) Tissue culture
plastic - Primaria plates from Falcon Co. were used
without additional preparation; 2) Type I collagen - 10
cm tissue culture dishes were coated with type I collagen
20 prepared from rat tail tendons. Michalopoulos , G. and n.
Pitot, Exp. Cellular Research , 94:70-78 (1575). Briefly,
collagen was solubilized in 0.1% acetic acid (3 mg/ml)
and applied to plates (1 ml/10 cm plate) which were
exposed to NH 3 vapors, air dried, sterilized by gamma
25 irradiation (10,000 rads) , and hydrated with media; 3)
Laminin - Purified laminin from Collaborative Research
Inc. (Waltham, MA) was applied to tissue culture plates
according to the recommendations of the manufacturer; 4)
Type IV collagen - 10 cm dishes coated with purified type
30 IV collagen were kindly provided by Dr. L.M. Reid (Albert
Einstein College of Medicine) .
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10
15
20
25
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EXAHPLE II Virus Preparation and Hepatocvte Infection
A helper-free amphotropic producer of the BAG virus
was provided by Dr. C. Cepko (Harvard). The retroviral
vector used to make this producer has been described by
Cepko and co-workers. Price, J., Proceedings of the
National Academy of Sciences, U.S.A. , 8^4:156-160 (1987).
It coexpressed beta-galactosidase from E. coli and the
bacterial gene that confers resistance to neomycin in
prokaryotes and to G418 in eukaryotes (neo) . The
producer was maintained in Dulbecco's modified Eagle's
Medium supplemented with 10% calf serum. Unconcentrated
viral stocks were prepared and titered as described by
Mann. Mann, R. et al, , Cell , 33:153-159 (19S3) . Titers
ranged from 1-4 x 1C 5 cfu/ml. Hepatocyte cultures were
infected for 12 hours with viral stocks (5 ml of viral
stock/ 10 cm plate of hepatocytes) containing 8 ug/ml of
Polybrene (Aldrich) . Transduction efficiency was
optimized with respect to the time of exposure to virus
and the matrix substrate on which the hepatocytes were
plated.
EXAMPLE III Assessment of Efficiency of Transduction
The efficiency of transduction was initially
assessed by directly measuring the integration of
provirus*
Southern Analysis
High-molecular-weight cellular DNA from transduced
cultures of hepatocytes was isolated as described
previously* Maniatis, T. et al. , Molecular Cloning: A
Laboratory Manual , Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1982). Aliquots (7.5 ug) were
digested with the restriction endonuclease Kpn I. Kpn I
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has recognition sites in the proviral long terminal
repeats; consequently , each integrated provirus will be
contained in a 6.9 Kb restriction fragment, irrespective
of the site of integration.
05 The restriction fragments were resolved by electro-
phoresis in 1% agarose gels and analyzed according to the
method of Southern using standard procedures and a probe
that is complementary to sequences unique to the provirus
(i.e., the neo gene). Maniatis, T. et al. , Molecular
10 Cloning: A Laboratory Manual , Cold Spring Harbor Labora-
tory, Cold Spring Harbor, N.Y. (1982) . The blot was
probed with the Bam/Hind III fragment of the neomycin
gene that was labelled to high specific activity with
32
P-dCTP using the random primer method. Feinberg, A. P.
15 and B. Vogelstein, Anals of Biochemistry , 132 : 6-13
(1983). The intensity of the resulting band on the auto-
radiograph is proportional to the number of proviral
integrants in the population.
Figure 4 shows the effect of extracellular matrix
20 used and of the time of infection on integration of
provirus in hepatocyte cultures. Panel A presents a
Southern blot of hepatocytes isolated from a single
collageriase perfusion which were cultured on 10 cm.
plates coated with one of several forms of matrix
25 substrata (type I collagen, laminin, type IV collagen,
and tissue culture plastic) and infected on days 1, 2, 3,
4, or 5. Infection was carried out using fresh prepara-
tions of viral stocks and plates were analyzed for copy
number of integrated provirus 48 hours after the infec-
30 tion was initiated.
A three day exposure of a Southern blot is shown. A
single band was visualized in each lane; the area of the
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autoradiograph containing this band is shown. Lanes 1-5
indicate the days that the cells were infected. The top
four series of bands represent hepatocytes cultured on
different forms of matrix: Col I - type I collagen,
05 Lam-laminin, Col IV - type IV collagen, and TCP-tissue
culture plastic. The bottom series of bands shows an
identical analysis of NIH3T3 cells infected with the same
viral stocks used to infect the hepatocyte cultures.
Hepatocytes on each matrix substrata exhibited a
10 l consistent pattern of susceptibility to transduction;
proviral integration increased from virtually undetect-
able on day 1 to maximal on days 2 or 3 , and subsequently
diminished to low levels by day 5. Maximal proviral
integration, which was essentially independent of matrix,
15 occurred when cultures were infected on day 2 for cells
on tissue culture plastic, or day 3 for cells on type I
collagen, laminin, or type IV collagen.
NIH3T3 cells were infected with the same viral
stocks used to infect hepatocytes (Fig. 1, panel A).
20 Southern analysis demonstrated little variation in the
titer of the viral stocks; the estimated proviral copy
number ranged from 0.5 to 0.7 copies/cell. This estimate
of copy number was based on a comparison with samples
with known quantities of standard plasmid (Fig. 4, panel
25 B) and the assumption that NIK3T3 cells are hypo-
tetraploid.
The samples with known quantities of standard
plasmid were made by mixing varying amounts (2 pg and 10
pg) of the purified BAG plasmid with 7.5 ug of uninfected
30 NIH3T3 DNA. Analysis showed a single band, which
comi grated with the bands shown in panel A. The data
from panels A and B were derived from a three day
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* exposure of the same Southern blot- It was estimated
that 2 pg and 10 pg of plasmid in 7.5 ug of NIH3T3 DNA
correlates to approximately 0.3 and 1.2 copies of
provirus/cell, respectively. The estimated copy number
03 of proviral integrants in maximally infected hepatocytes
(e.g., Figure 4, Lam, lane 3) is approximately 0.2
copies/cell, assuming that the DNA content of NIH3T3
cells is equal to that of hepatocytes. This assumption
is probably valid since the majority of hepatocytes in
Iff culture are either tetraploid or octaploid. Tomita, Y.,
et al. , Exp. Cell Res. , 135:263-370 (1981).
Cytochemical and Immunocytochemical Procedures
A series of liver-specific cytochemical and immuno-
chemical stains was used to document the cellular
15 composition of the hepatocyte cultures. All analyses
were performed on three day old cultures of hepatocytes
plated on type I collagen.
Cells infected with the BAG virus constitutive ly
produce high levels of cytoplasmic beta-galactosidase.
20 Price, J. et al. , Proceedings of the National Academy of
Sciences, U.S.A. , 84:156-160 (1987). Activity of
beta-galactosidase was detected in situ with the sub-
strate 5-bromo-4-chloro-3-indolyl-D-galactosidase, which
forms a blue precipitate in infected cells. Price, J. et
25 al., Proceedings of the National Academy of Sciences f
U.S.A. , 84; 156-160 (1987).
Duplicate cultures of infected hepatocytes were
analyzed in situ for retrovirus-transduction (and expres-
sion) by the cytochemical stain for beta-galactosidase.
30 Price, J. et al. , Proceedings of the National Academy of
Sciences, U.S.A. , 84:156-160 (1987). This procedure
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specifically labels cells that express viral directed
beta-galactosidase? endogenous beta-galactosidase is
not detected. Figure 5 (panels A-E) shows cytochemical
, localization of beta-galactosidase activity in transduced
OS cultures of hepatocytes and NIH3T3 cells. Panels A-E
show hepatocyte cultures plated on type I collagen and
infected on days 1, 2, 3, 4, or 5 (panels. a-e f respec-
tively) . Panel f shows a population of NIH3T3 cells
which had been infected with the same viral stocks used
10 to infect hepatocytes on day 3 .
Results showed that labeled cells are often found in
groups of 2, probably representing infection and integra-
tion into a dividing cell with labeling of the two
daughter cells» In addition, expression of beta-
15 galactosidase, as determined by the intensity of stain-
ing, is quite variable, but tends to be consistent within
members of a pair of labeled cells (e.g., see panel C) .
The -efficiency of transduction, as measured cytochemi-
cally, exhibited the same dependence on time in culture
20 as was demonstrated by Southern analysis. The fraction
of labeled cells increased from less than 1% in cultures
infected on day 1 to approximately 25% when infected on
day 3; the transduction efficiency dropped dramatically
in cultures infected on the next 2 days (days 4 and 5).
25 Similar analysis of NIH3T3 cells infected with the
same viral stock used to infect day 3 hepatocytes indi-
cated that approximately 50% of the cells were labelled.
This is consistent with the estimated efficiency of
transduction based on Southern blot analysis (Figure 4,
30 panel A) .
Imm\inocytochemical localization of UDP-glucuronosyl-
transferase and asialoglycoprotein receptor was performed
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in the culture dishes using horseradish peroxidase
conjugated to protein A (from Staphylococcal aureus ) and
diaminobenzidine cytochemistry at pH 7.4 to detect
peroxidase activity. Novikoff, P.M. et al« , Journal of
05 Cell Biology , 92=1559-1565 ( 1983 > * Monospecific IgG to
rat UDP-glucuronosyltransf erase was purified from rabbit
antiserum. Chowdhury et al. used immunocytochemical
techniques to determine the distribution of UDP-
glucuronosyltransf erase in the liver. This membrane-
10 bound enzyme is present exclusively in hepatocytes and is
localized to the endoplasmic reticulum and nuclear
membrane. Chowdhury, J.R. et al. , Proceedings of the
National Academy of Sciences, U.S.A. , 82:2990-2994
(1985). Dr. R. Stockert -(Albert Einstein College of
15 Medicine) kindly provided monospecific antibody to rat
asialoglycoprotein receptor. Controls for the immuno-
cytochenical experiments included exposure of cells to
pre- immune rabbit antisera, followed by identical
procedures as those employed for specific rabbit
20 antibody. Immunocytochemical analysis of cultured
hepatocytes using the monospecific polyclonal antibody to
UDP-glucuronosyltransferase shows reaction product
distributed in cytoplasmic clumps and at the periphery of
the nucleus in greater than 95% of the cells (Figure 5,
25 panel G) ; these reactive sites correspond to the endo-
plasmic reticulum and the nuclear envelope, respectively.
No reaction product is seen in experiments performed with
preimmune rabbit IgG (Figure 5, panel i) .
Asialoglycoprotein receptor
30 This well described receptor is specifically
expressed in hepatocytes. Immunocytochemical analysis in
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rat liver localizes this receptor to a domain of the
plasma membrane which borders the sinusoids; under light
microscopy the receptor is seen at the perimeter of the
hepatocyte along its sinusoidal face. Genze, J.E. et
05 al. r Journal of Cellular Biology , 921:867-870 (1982);
Matsuura, S. et al, r Journal of Cellular Biology ,
95:864-875 (1982). The level of asialoglycoprotein
receptor decreases in culture; however, it is still
demonstrated in virtually all cells of a three-day-old
10 hepatocyte culture . Reaction product is seen as a dense
line in focal regions of the hepatocyte periphery (Figure
5, panel H, in which results of localization with a mono-
specific rabbit antibody are shown) . This characteristic
staining is absent in experiments with control (pre-
15 immune) rabbit serum (Figure 5, panel i) .
Glucose-6-phosphatase
This glycolytic enzyme is a well recognized cy to-
chemical marker for hepatocytes. It can be detected in
virtually all hepatocytes of liver sections. However,
20 there is marked regional variation in enzyme activity;
the greatest activity is found in the periportal region.
Sasse, D., "Regulation of Hepatic Metabolism and Intra-
and Intercellular Compartmentalization, eds. Thurman,
R.G. , Kauffman, F. C. and Jungermann, K. (Plenum Press,
25 NYC) , pp. 57-86 (1986) .
Glucose-6-phosphatase activity was detected in
three-day-old hepatocyte cultures by the lead phosphate
enzyme cytochemical procedure. Wachstein, M. and E.
Meisel, J. Histochem. Cvtochem. , £:592 -(1956).
30 Characteristic brown/black cytoplasmic staining was
seen in greater than 95% of the cells (Figure 5, panel
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J) . As expected, there was marked cell-to-cell variation
in enzyme activity. Activity was not detected in pure
cultures of nonparenchymal cells such as fibroblast.
Peroxisomes
05 The method of Novikoff et al. was used to visxialize
. the distribution of peroxisomes in hepatocyte cultures.
Novitoff, A.. B. et al . , J. Histochemistry and Cyto-
chemistry , 20:1006-1023 (1972). These small cytoplasmic
structures (approximately 0.5 microns in diameter) are
10 found specifically in hepatocytes (in the context of the
liver) and are visualized by cytochemical staining for
catalase. DeDuve, C. et al. f Physiol. Rev. , 46 : 323-357
(1966).
Greater than 95% of the cells in the culture tested
15 demonstrated numerous catalase-pcsitive peroxisomes,
v/hich appeared as dot-like structures distributed
randomly throughout the cytoplasm (Figure 5, panel K) .
Peroxisomes were not detected when pure cultures of
nonparenchymal cells (e.g., fibroblasts) were analyzed.
20 EXAMPLE IV Retrovirus-Mediated Gene Transfer to
Correct LDLR Deficiency
Isolation, Maintenance and Infection of Hepatocvtes
Newborn New Zealand white (NZV7) rabbits, and
Watanable heritable hyperlipidemic (WHHL) rabbits (3-5
25 days old weighing 50-80 gms) were used as the source of
hepatocytes. The WHHL rabbit has been shown to be
deficient in functional LDLR activity due to an in-frame
deletion of a portion of the LDLR structural gene.
Yamamoto, T. et al. , Science , 232 ; 1230-1237 (1986) . NZW
30 rabbits have been used as controls in most previous
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studies of the WHHL rabbit. Newborn WUHL rabbits were
derived from matings between homozygous deficient males
and females and were kindly provided by Dr. Knapka (NIE) .
Four WHKL rabbits from 2 litters (2 rabbits /litter) were
05 used in these studies (named WHHL 1-4) . Newborn NZW
rabbits were purchased from Pine Acres Rabbitry (West
Brattleboro f Vermont) . Hepatocytes were prepared using a
modification of the procedure of Berry and Friend with
the perfusion ..mixture of Leffert. Berry , M.N. and D.S.
1Q Friend, Journal of Cell Biology , 42:506-520 (1969).
Leffert, H.L. et aL , Methods in Enzymoloqy , 58:536-544
(197 9) . Collagenase perfusions were performed retrograde
as described by Clayton and Darnell for the preparation
of adult mouse hepatocytes. Clayton, D.F. and J.E.
15 Darnell, Jr., Molecular Cell Biology , 2 :1552 " 1561 (1983).-
Cells were plated at a density of 3-4 x 10 4 cells/cm 2
onto Primaria plates (Falcon Co.) in hormonally defined
media supplemented with 10% fetal bovine serum; 4-6 hours
later the media was replaced with fresh hormonally
20 defined media which was subsequently changed every 24
hours during the duration of the experiment. Enat, R. et
al» / Proceedings of the National Academy of Sciences,
USA , 81rl411-1415 (1984).
Hepatocyte cultures were infected for 12 hours with
25 viral stocks (5 ml/10 cm plate) containing Polybrene (8
ug/ml) . Unconcentrated viral stocks were prepared from
the producer cells as described above.
DNA and RNA Analysis . High-molecular-weight genomic
DNA was isolated and analyzed for integration of proviral
30 sequences. Total cellular RNA was prepared using a
guanidine thiocyanate procedure, fractionated in
formaldehyde/agarose gells and transferred to
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nitrocellulose paper. Chirgwin, J.M. et al. ,
Biochemistry , 1J3: 5294-5299 (1980). Northern and Southern
blots were probed with a 1.9 kb LDLR cDNA fragment (Hind
III to Eco RI fragment of pTSl) that was labeled to high
0.5 specific activity with 3z P-dCTP using the random primer
method. Feinberg, A. P. and B. Vcgelstein, Anal.
Biochem. f 132 : 6-13 (1984). Northern blots were stripped
and reprobed with a cDNA probe for human gamma actin
(Hind III to Bam HI fragment of pHF-1) . Gunning, P. et
1Q al^, Molecular Cell Biology , 3:787-795 (1983).
Cytochemical Analyses . Hepatocytes cultures
infected with the BAG virus were analyzed for expression
of viral directed beta-galactosidase using a cytochemical
stain that forms a blue precipitate in the cytoplasm of
15 transduced cells. Price, J. et al. , Proceedings of the -
National Academy of Sciences, USA , 84:156-160 (1987).
Glucose-6-phosphatase activity was detected by the lead
phosphate enzyme cytochemical procedure. VTachstein, M.
and E. Meisel, J. Histochem. Cvtochem. , £:592 (1956) .
20 Cultures were analyzed for the presence of LDLR or the
receptor for acetylated LDL (AcLDL) by incubating the
cultures in hormonally defined media containing
fluorescent labeled LDL or AcLDL (labeled with 1,1'-
dioctadecyl-3 , 3 , 3 1 3 * , -tetramethy lindocarbacyanine
25 perchlorate - hereafter abbreviated as Dil - at 10 ug/ml,
and obtained from Biomedical Tech. Inc., Stoughton, MA)
for 6-8 hours, followed by three rinses with phosphate
buffered saline and fixation in phosphate buffered saline
containing 0.5% gluteraldehyde. Pitos, R.E. et al. ,
30 Arteriosclerosis , li 177-185 (1981). Voyta, J.C. et al. ,
Journal of Cellular Biology, _99:81A (1984). Uptake of
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the fluorescinated reagents was visualized in situ using
an inverted Leitz fluorescent microscope.
Assay of LDL Degradation . Five-day-old cultures of .
hepatocytes plated in 35 rani dishes were assayed for
125
05; degradation of I -LDL (10 ug/ml r 0.15 uCi/ug, obtained
from Biomedical Tech. , Inc. , Stoughton r MA) using the
procedure described by Goldstein, Basu and Brown.
Goldstein, J*L. et al. , Methods in Enzymoloqy , 98:241-260
(1983>.
10 Generation of Recombinant Retroviruses Encoding
Human LDLR
Four different retroviral vectors were tested; the
proviral components of these vectors are presented in
Figure 3. Each vector differs in the transcriptional
15 elements used to drive the expression of LDLR: LTR-LDLR -
viral long terminal repeat sequences (LTR) ; BA-LDLR
contains a 267 bp segment of the chicken beta-act in gene
(BA) extending from -266 to +1 . H4-LDLR contains a 704
bp segment of the histone H4 gene (H4) extending from
20 -696 to +8. TK-LDLR contains a 256 bp segment of the
thymidine kinase gene of herpes simplex virus (TK) ,
extending from -200 to +56. Plasmid sequences of
LTR-LDLR were derived from the 7.2 Kb Bam HI to Cla I
fragment of DC1 with the following modification:
25 sequences spanning the Nhe I to Xba I sites of the 3 f
Moloney murine leukemia virus (Mo-MLV) LTR (nuc. 7846 to
8113) were replaced with homologous sequences from the
LTR of the myeloproliferative sarcoma virus (represented
by darkened area) . Korman, A.J. et al. f Proceedings of
30 the National Academy of Sciences f USA , 84r2150-2154
(1987); Van Beveren, C. et al. , In: RITA Tumor Viruses
(2nd edition) , Weiss, R. et al. (ed.) , Cold Spring Harbor
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Laboratory, pp. 766-783 (1985) ; Stacey, A. et aL ,
Journal of Virology , 50:725-732 (1984).
The backbone structure of plasmids BA-LDLR, H4-LDLR,
and TK-LDLR (including the 5' -LTR, flanking mouse genomic
05 DNA, pBR322 sequences, and 3 ' -LTR with contiguous pro-
viral sequence to the Cla I site at nucleotide 7674) was
derived from DOl, with the exception that sequences
containing the viral enhanced elements of the 3 1 LTR
(from the Pvu II site at nucleotide 7933 to the Xba I
10 site at nucleotide 8111) were deleted (indicated by the
inverted triangle) . This was done to reduce the amount
of viral transcription after reverse transcription and
integration of the recombinant provirus. These vectors
also contained additional Mo-MLV sequence between the 5 '
15 LTR and the internal promoters. The additional sequence -
was derived from wild type Mo-IiLV (from nucleotide 146 at
the border of U5 to the Xho I site in the gag coding
region at nucleotide 1560) with the exception that a Sac
II linker was inserted at the Kae III site at nucleotide
20 624. (This additional sequence is noted as gag in the
figure) . In each case LDLR coding sequences were derived
from a 2.6 kb Hind III fragment of plasmid pTZl which
contains a full-length LDLR cDNA insert (kindly provided
by Drs. D. Russell , J. Goldstein and M. Brown) . S.D.
25 indicates splice donor site; arrows under each vector
show the sites of transcriptional initiation.
Virus producing cell lines for the vector BA-LDLR ,
H4-LDLR, and TK-LDLR were made by transfecting the
particular plasmid DNA with pSV2-Neo into the amphotropic
30 packaging cell line Psi-Crip, as described previously.
Cone, R.D. et al. , Molecular and Cellular Biology , 7:887-
897 (1987); Mulligan, R.C. and P. Berg, Science ,
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209 ;1422-1427 (1980). Psi-Crip is a modified type of
Psi-am packaging cell line which provides cells with the
functions of Psi-am (e.g., gag , pol , and env ) in two
components. That is, the gag - pol function is provided in
Q5 z j the form of one integrated provirus and the env function
is produced from a separate provirus. The host range is
amphotropic .
G418 resistant colonies of cells v/ere expanded and
tested for production of virus that transmitted the
10* correct proviral structure. This was done by harvesting
sxipernatants from the producer cells, infecting KIK3T3
cells, and analyzing the infected population for
integrated provirus by Southern analysis.
High titer amphotropic producers of the LTR-LDLR
15 vector were obtained using a 2-step procedure. First,
high titer ecotropic producers were made by cotransfec-
ticn of LTR-LDLR with pSV2-tteo into the Psi-2 packaging
cell line as described above. Ilann, R. et al. , Cell ,
32*153-159 (1983). Psi-Crip cells were then infected
20 with virus harvested from the Psi-2 producer and subse-
quently split into 10 cm plates at clonal densities.
Individual clones were isolated and analyzed for the
production of high titer amphotropic virus as described
above. Virus-producing cell lines transmitting the
25 highest number of proviral copies to recipient cells were
chosen for this study. All virus producing cell lines
were maintained in culture for 4-6 weeks prior to their
use in order to test for the presence of helper virus.
None of the cell lines yielded any detectable helper
30 virus nor transferred the packaging functions. A NIH3T3
cell line, designated 7-35, producing amphotropic virus
that expresses the gene for human LDL recepter has been
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deposited (February 3, 19 88) under the terms of the
Budapest Treaty r with the American Type Culture
Collection (Rockville, I ID) under accession number CRL
9635.
05 Transfer and Expression of LDLB in Hepatocytes
Cells used for the infection studies were hepato-
cytes isolated from 3 NZW rabbits and 4 V7HHL rabbits as
described above. Collagenase perfusions routinely
produced 40-80 x 10 6 cells/animal with greater than 90%
10 viability. Cells plated at subconfluent densities formed
aggregates (5-20 cells/aggregate) that covered approxi-
mately 20% of the dish when visualized 6 hours after
plating . The primary cultures underwent marked pro-
liferation after 36 hours in culture achieving confluence
15 by day 3 or 4.
To document the cellular composition of the
cultures, mock infected WHRL hepatocytes cultured for 5
days were analyzed in several ways* First, the cells
were stained for glucose-6-phosphatase f as described
20 above, to determine the number of hepatocytes in the
cultures. Glucose-6 -phosphatase is a specific marker for
hepatocytes in sections of liver and in hepatocyte
cultures. More than 95% of the cells had the brown
cytoplasmic staining characteristic of hepatocytes. No
25 staining was detectable in pure cultures of fibroblasts
or endothelial cells.
In addition, the cultures were analyzed for the
presence of endothelial cells and Kupffer cells, since
these nonparenchymal cells are abundant in the intact
30 liver and could potentially contaminate the primary
cultures. Sinusoidal and capillary endothelial cells as
well as Kupffer cells express high levels of the receptor
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for AcLDL and can be identified in mixed cultures by
their selective uptake of Dil- AcLDL* Analysis of the
cultured hepatocytes for Dil- AcLDL uptake revealed that
approximately 1 in 50 cells v/ere fluorescent. The uptake
05 of Dil -AcLDL by the rare contaminating cells in the
hepatocyte cultures was equivalent to that observed in
pure secondary cultures of endothelial cells derived from
bovine aorta.
To optimize the conditions for infection of the .
10 hepatocytes, a high titer araphotropic virus (BAG) en-
coding coli beta-galactosidase was used. Cells
transduced by the BAG virus can be detected by a simple
cytochemical reaction that stains the cell's cytoplasm
blue. Optimal transduction of V7HHL hepatocytes was
15 achieved when the cells were plated at subconfluent
density and exposed to virus 36 hours after the initial
plating. The matrix substrata had little effect on
transduction efficiency .
Having optimized the conditions for infection, NZW
20 and WHHL hepatocytes were infected with the four. dif-
ferent LDLR virus preparations 2 days after being placed
in culture and were analyzed for gene transfer and LDLR
expression on day 5» Integration of the recombinant
proviral sequences into the cellular DNA isolated from
25 infected hepatocytes was detected by Southern blot
analysis (Figure 6) . DNA from transduced hepatocytes was
digested with Kpn I and analyzed by the method of
Southern using the LDLR cDNA as a probe. Kpn I has
unique recognition sites in the LTR sequences; conse-
30 quently, each integrated provirus should yield a common
restriction fragment irrespective of the site of
integration. Each virus producing cell line efficiently
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transraitted proviral sequences without rearrangement in
hepatocytes from both NZW and WHHL rabbits. The relative
copy number of integrated provirus varied from a maximum
of 1-2 copies/cell for cultures infected with the H4-LDLR
05 virus to a minimum of 0.1 to 0.2 copies/cell for cultures
infected with the LTR-LDLR virus. This efficiency of
infection was approximately 50% of that achieved in
murine fibroblastic cells infected with the sane virus
preparations.
10 Additional experiments were performed to show that
the viral DNA detected in Figure 6 was integrated into
hepatocyte DNA. DNAs from transduced . hepatocytes were
digested with Eco RI (a restriction enzyme that has a
single site in the proviral DNA) and subjected to
15 Southern analysis using an LDLR probe. If the viral DNA
existed as an integrated provirus no distinct Eco RI
fragments should be detected because the outer borders of
these fragments are located in flanking DNA and therefore
are heterogenous. In fact, no Eco RI fragments were
20 detected when this analysis was done, suggesting that the
majority of viral DNA was integrated into hepatocyte
chromosomal DNA.
Transduced cultures were first analyzed for LDLR
expression by Northern analysis (Figure 7) . A faint band
25 with an apparent molecular size equal to 3.5 Fvb was
detected in mock infected cultures (Figure 7) . This
band, which probably represents endogenous LDLR RNA, was
consistently more intense in WHHL cultures than in. NZW
cultures. The predominant RNA species in cultures
30 infected with BA-LDLR, H4-LDLR, and TK-LDLR were the
transcripts initiated at the internal promoter. The
relative abundance of these RNAs consistently varied in
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vector-dependent manner as follows: BA-LDLR H4-LDLR
TK-LDLR putative endogenous signal. As expected, very
little transcription initiated from the LTR of these
vectors was detected since the enhancer deletion which is
05 present in the 3 1 LTR of the starting plasmid is
transferred to the 5 T LTR during proviral passage into
hepatocytes. Cultures infected with the LTR-LDLR virus
produced a single very intense band representing a
transcript initiated at the LTR. All blots were stripped
Id and reprobed with a human gamma-action cDNA probe to
control for variation in the amount of RNA that was
loaded- There was no detectable variation in the
intensity of the gamma-action band suggesting that equal
quantities of undegraded RITA were loaded,
15 Biochemical activity of the exogenous LDLR was
.assessed in situ by visualizing the uptake of LDL;
transduced cultures of hepatocytes were incubated with
Dil-LDL and viewed by fluorescent microscopy. Mock
infected HZW rabbits exhibited a uniformly high level of
20 fluorescence in all cells; mock infected WHHL hepatocytes
shewed very little fluorescence. WHHL hepatocytes
infected with the LTR-LDLR virus had the greatest amount
of LDL uptake with approximately 20% of the cells showing
high levels of fluorescence. BA-LDLR infected V7HHL
25 hepatocytes demonstrated a population of cells with,
moderate activity; H4-LDLR infected WHHL hepatocytes
showed a low level of activity in virtually all cells.
The activity of LDLR in TK-LDLR infected cells was barely
over background. The estimate of transduction efficiency
30 based on this in situ assay for LDLR activity agrees with
that measured by Southern analysis (e.g., V7HKL
hepatocytes infected with the LTR-LDLR virus showed
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fluorescence in approximately 20% of cells while Southern
analysis detected a copy number of integrated proviral
sequences equal to approximately 0.2).
- Transduced hepatocytes were also analyzed for
125
05 degradation of I-LDL in an attempt to quantify the
amount of human LDLR expressed. These data are sum-
marized in the Table. Activity of LDLR was greatest in
hepatocytes infected with the LTR-LDLR virus: hepato-
cytes from a NZV7 rabbit showed an increase in LDLR
10 activity from 170 ng/mg/5hrs in mock infected cells to
274 ng/mg/5hrs in transduced cells, while cells from WHHL
rabbits exhibited an increase in activity from 30-40
ng/mg/5hrs in mock infected cells to 155 (WHHL 1) and 84
(V7KKL 3) ng/mg/5hrs in transduced cells. The level of
15 LDLR activity in. LTR-LDLR transduced hepatocytes is
approximately 700 ng/mg/5hrs (4 fold greater than the
activity of the endogenous receptor in MZV7 rabbits) when
corrected for the actual number of cells that were
transduced. Dzierzak, E.A. et al. , Nature , 331:35-41
20 (1987). Hepatocytes infected with viruses that express
LDLR from a transcript driven by an internal promoter
(i.e., BA-LDLR, H4-LDLR, and TK-LDLR) exhibited little to
125
modest increases in I-LDL degradation.
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Table
L25
Quantitative analysis of I-1DL degradation in transduced
heoatocvtes^
05.
125 2
I-IDL Degradation fng/mg orotein/5h)
Virus
VHHL-1
WHHL- 3
mock
170+8 3
3 8+5 3
BA-LDLR
201+10
42+3
R4-LDLR
194+9
30 4
47+3
TrC-LDLH
188+17
44+8
LTR-LDLR
274+6
155 4
84+13
10 ^Degradation races were also measured in the presence of 50 fold excess of
unlabeled LDL. Under these conditions NZW hepacocytes (mock infected and
transduced) had degradation races that ranged from 50 to 60 ng/mg/5 hrs
while WHHL hepacocytes had degradation rates that ranged from 10 to 20
*S/mg/5 hrs.
IE* Analyses were performed on selected cultures of one NZW rabbit and two WHKL
rabbits (WHHL 1 and WHHL 3).
Represents mean +1 S.D. (N-3 for WHHL 3 and N-4 for NZW) .
4
Single determinations-
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EXAMPLE V Introduction of Genetically Modified
Hepatocytes into Rabbits
The technique described by Demetriou et al. was used
to develop methods to restore LDL recepter function in
05 the WHHL rabbit. Demetriou, A. A. et al. , Science , 233 :
1190-1192 (1986) . Briefly, allogenic hepatocytes with
normal LDL recepter function were isolated from New
Zealand white (NZW) rabbits by collagenase perfusion.
The isolated hepatocytes were bound to collagen coated
10 dextran microcarrier beads and injected intraperitoneally
g
(IP) into WHHL rabbits (2 >: 10 cells/ rabbits) . As a
control for nonspecific effects of the intervention on
serum cholesterol, hepatocytes from outbred WHHL rabbits
bound to microcarrier beads were injected IP into another
15 group of WHHL rabbits.
Serum was subsequently obtained and analyzed at
various times for serum cholesterol levels. In the group
of WHHL rabbits injected with NZV: hepatocytes, serum
cholesterol progressively declined by 25 +/- 4% (N=4)
20 over 3 to 4 days, and subsequently increased to baseline
levels by day 9. In the control group, serum
cholesterol levels actually increased transiently to 114
+/- 4% (N=2) of control and returned to baseline levels
by day 7.
25 A second set of similar experiments was performed in
which the hepatocytes from the NZW rabbits were injected
into the portal veins of WHHL rabbits. The serum
cholesterol levels progressively declined by 25 +/- 4%
(N=3) for four days after injection and subsequently
30 increased to baseline levels by day 11.
In another experiment, hepatocytes from a WHHL
rabbit were isolated by collagenase perfusion for in vivo
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studies. The hepatocytes were plated as primary
cultures , infected with either the LTR-LDLR recombinant
retrovirus or mock infected (See Example IV) . The
hepatocytes were harvested and injected into the portal
05t vein of WHHL rabbits. The animal transplanted with
mock- infected cells demonstrated a transient increase in
cholesterol to a 115 +/- 6% (17=2) , while the animal
transplanted with LTF-LDL infected cells demonstrated a
decrease in serum cholesterol by 30% over a 3 day period.
10 EXAMPLE VI Expression of human parathyroid hormone by
rat hepatocvtes
Hepatocytes were isolated from a Kistar rat, as
described in Example I. Cells were plated at a density
of 10 x 10 6 cells onto a bacteriologic plate (10 cm) with.
15 160 mg of cytodex beads and inoculated with hormonally
defined medium (HDM) with 10% fetal calf serum. After 90
minutes, the media was replaced with fresh HDM. On day
three, the plate was inoculated with viral supernatant
from an amphotropic PTE producer (prepared as described
20 in Example II) . After 12 hours, the cells were trans-
planted into an analbumineraic rat (approximately 200 gm
intraperitoneally in approximately 5 ml* phosphate
buffered saline) .
Serum was subsequently obtained and analyzed at
25 various times (see Figure 8) for human PTH, using the
commercially-available Nichols radioimmunoassay
procedure. Serum was analyzed for rat albumin using
Western blot analysis. Pvesults are presented in Figure
8.
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Equivalents
Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation,
many equivalents to the specific embodiments of" the
05 invention described specifically herein. Such equiva-
lents are intended to be encompassed in the scope of the
following claims.
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